•The types and applications of main organic acids were overviewed.•Production of organic acids by protein and metabolic engineering strategies were summarized.•The future prospects for organic acids production using systems and synthetic biology were discussed.Organic acids are natural metabolites of living organisms. They have been widely applied in the food, pharmaceutical, and bio-based materials industries. In recent years, biotechnological routes to organic acids production from renewable raw materials have been regarded as very promising approaches. In this review, we provide an overview of current developments in the production of organic acids using protein and metabolic engineering strategies. The organic acids include propionic acid, pyruvate, itaconic acid, succinic acid, fumaric acid, malic acid and citric acid. We also expect that rapid developments in the fields of systems biology and synthetic biology will accelerate protein and metabolic engineering for microbial organic acid production in the future.

•Overexpression of native pyruvate carboxylase and malate dehydrogenase in the rTCA pathway.•Construction of oxaloacetate anaplerotic reaction.•Overexpression of a C4-dicarboxylate transporter from A. oryzae and an L-malate permease from Schizosaccharomyces pombe.•Overexpression of the 6-phosphofructokinase which was identified as a potential limiting step for L-malate synthesis.Aspergillus oryzae finds wide application in the food, feed, and wine industries, and is an excellent cell factory platform for production of organic acids. In this work, we achieved the overproduction of L-malate by rewiring the reductive tricarboxylic acid (rTCA) pathway and L-malate transport pathway of A. oryzae NRRL 3488. First, overexpression of native pyruvate carboxylase and malate dehydrogenase in the rTCA pathway improved the L-malate titer from 26.1 g L−1 to 42.3 g L−1 in shake flask culture. Then, the oxaloacetate anaplerotic reaction was constructed by heterologous expression of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase from Escherichia coli, increasing the L-malate titer to 58.5 g L−1. Next, the export of L-malate from the cytoplasm to the external medium was strengthened by overexpression of a C4-dicarboxylate transporter gene from A. oryzae and an L-malate permease gene from Schizosaccharomyces pombe, improving the L-malate titer from 58.5 g L−1 to 89.5 g L−1. Lastly, guided by transcription analysis of the expression profile of key genes related to L-malate synthesis, the 6-phosphofructokinase encoded by the pfk gene was identified as a potential limiting step for L-malate synthesis. Overexpression of pfk with the strong sodM promoter increased the L-malate titer to 93.2 g L−1. The final engineered A. oryzae strain produced 165 g L−1 L-malate with a productivity of 1.38 g L−1 h−1 in 3-L fed-batch culture. Overall, we constructed an efficient L-malate producer by rewiring the rTCA pathway and L-malate transport pathway of A. oryzae NRRL 3488, and the engineering strategy adopted here may be useful for the construction of A. oryzae cell factories to produce other organic acids.

•Membrane proteomes of E. coli under different phenylpropanoids were analyzed.•16 differentially expressed membrane proteins were identified by MALDI-TOF/TOF MS.•Effect of 14 proteins on E. coli were studied by gene overexpression and silencing.Phenylpropanoids are phytochemicals produced by some plants and possess a wide variety of biological activities. These compounds exist in plants in low amounts. Production of them in genetically engineered microorganisms has many advantages. A majority of functional phenylpropanoids are toxic to microbial hosts. Export of these compounds may relieve the cellular toxicity and increase the yield. However, proteins and mechanisms involved in phenylpropanoids transport and tolerance remain poorly understood. In this study, 16 membrane proteins that were differentially expressed in Escherichia coli in response to three typical phenylpropanoids (resveratrol, naringenin and rutin) were identified using a membrane proteomics approach. These proteins included outer membrane proteins OmpA, OmpF, OmpW, FadL, TolC, LamB, and YaeT, peripheral membrane proteins AtpD, AtpH, YgaU, OppA, MalK, and MalE, and cytoplasmic membrane proteins OppD, PotG, and ManX. Functions of these proteins were determined by using gene overexpression and silencing. The results suggest that OmpA and FadL may play important roles in the transmembrane export of phenylpropanoids in E. coli. LamB, MalE, MalK and ManX may participate in phenylpropanoid uptake. The role of YgaU in enhancing the tolerance to phenylpropanoids remains to be determined. These results may assist the engineering of microorganisms with enhanced phenylpropanoid producing capabilities.Biological significancePhenylpropanoids are phytochemicals produced by some plants and possess a wide variety of biological activities. Both the tolerance and the transport of phenylpropanoids play important roles in systematic metabolic engineering of microorganisms to produce these phytochemicals. Both specific and non-specific transporters are essential for these functions but remain poorly understood. This research utilized membrane proteomics to identify E. coli BL21 (DE3) membrane proteins that may be involved in phenylpropanoid transport and tolerance. These results may facilitate the construction of more efficient microbial phenylpropanoid producers through genetic engineering of membrane transporter proteins.

•The specificity of CGTase towards maltose was improved by site-saturation engineering of lysine 47.•The mechanism for enhanced specificity was clarified by structure modeling of the CGTase.•Enzymatic synthesis of AA-2G with maltose as glycosyl donor was optimized.•The obtained CGTase mutants have great potential in the large-scale production of AA-2G.In this work, the specificity of cyclodextrin glycosyltransferase (CGTase) of Paenibacillus macerans towards maltose was improved by the site-saturation engineering of lysine 47, and the enzymatic synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) with l-ascorbic acid and maltose as substrates was optimized. Compared to the AA-2G yield of the wild-type CGTase, that of the mutants K47F (lysine→phenylalanine), K47P (lysine→proline), and K47Y (lysine→tyrosine) was increased by 17.1%, 32.9%, and 21.1%, respectively. Under the optimal transformation conditions (pH 6.5, temperature 36 °C, the mass ratio of l-ascorbic acid to maltose 1:1), the highest AA-2G titer by the K47P reached 1.12 g/L, which was 1.32-fold of that (0.85 g/L) obtained by the wild-type CGTase. The reaction kinetics analysis confirmed the enhanced maltose specificity of the mutants K47F, K47P, and K47Y. It was also found that compared to the wild-type CGTase, the three mutants had relatively lower cyclization activities and higher disproportionation activities, which was favorable for AA-2G synthesis. As revealed by the interaction structure model of CGTase with substrate, the enhancement of maltose specificity may be due to the removal of hydrogen bonding interactions between the side chain of residue 47 and the sugar at −3 subsite. The obtained mutant CGTases, especially the K47P, has a great potential in the large-scale production of AA-2G with maltose as a cheap and soluble substrate.

•This is the first report about the biosynthesis of homoeriodictyol by enzymatic transformation.•The flavone 3′-O-methyltransferase ROMT-9 gene from rice was synthesized and expressed in Y. lipolytica.•The ROMT-9 was purified and characterized in terms of optimal pH, temperature, and catalytic kinetics.•The maximum amount of homoeriodictyol reached 110 mg/L with a transformation ratio of 52.4% at 16 h.•The biotransformation has less environmental pollution compared with the traditional chemical synthesis.In this work, we attempted to synthesize homoeriodictyol by transferring one methyl group of S-adenosyl-l-methionine (SAM) to eriodictyol using flavone 3′-O-methyltransferase ROMT-9, which was produced by recombinant Yarrowia lipolytica. Specifically, the ROMT-9 gene from rice was synthesized and cloned into the multi-copy integrative vector pINA1297, and was further expressed in Y. lipolytica with a growth phase-dependent constitutive promoter hp4d. The highest ROMT-9 activity reached 5.53 U/L after 4 days of culture in shake flask. The optimal pH and temperature of the purified ROMT-9 were 8.0 and 37 °C, respectively. The purified enzyme was stable up to 40 °C, and retained more than 80% of its maximal activity between pH 6.5 and 9.0. The recombinant ROMT-9 did not require Mg2+ for catalysis, while was completely inhibited in the presence of 5 mM Zn2+, Cu2+, Ba2+, Al3+, or Ni2+. The purified ROMT-9 was used to synthesize homoeriodictyol, and the maximal transformation ratio reached 52.4% at 16 h under the following conditions: eriodictyol 0.2 g/L, ROMT-9 0.16 g/L, SAM 0.2 g/L, CH3OH 6% (v/v), temperature 37 °C, and pH 8.0. This work provides an alternative strategy for efficient synthesis of homoeriodictyol and compared to the traditional plant extraction or chemical synthesis, the biotransformation approach generates less environmental pollution and has a great potential for the sustainable production of homoeriodictyol.

•The subsite-3 of CGTase was systematically engineered to improve maltodextrin specificity.•The AA-2G yield was improved by 85.8% by a quadruple CGTase mutant K47L/Y89F/N94P/D196Y.•The reaction kinetics of all the CGTase mutants was systematically analyzed and modeled.•The pH stability and thermal stability of all the mutants were analyzed.•The mechanism for the enhanced maltodextrin specificity was explored by structure modeling.In this work, the subsite-3 of cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans was engineered to improve maltodextrin specificity for 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) synthesis. Specifically, the site-saturation mutagenesis of tyrosine 89, asparagine 94, aspartic acid 196, and aspartic acid 372 in subsite-3 was separately performed, and three mutants Y89F (tyrosine → phenylalanine), N94P (asparagine → proline), and D196Y (aspartic acid → tyrosine) produced higher AA-2G titer than the wild-type and the other mutants. Previously, we found the mutant K47L (lysine → leucine) also had a higher maltodextrin specificity. Therefore, the four mutants K47L, Y89F, N94P, and D196Y were further used to construct the double, triple, and quadruple mutations. Among the 11 combinational mutants, the quadruple mutant K47L/Y89F/N94P/D196Y produced the highest AA-2G titer of 2.23 g/L, which was increased by 85.8% compared to that produced by the wild-type CGTase. The reaction kinetics of all the mutants were modeled, and the pH and thermal stabilities of all the mutants were analyzed. The structure modeling indicated that the enhanced maltodextrin specificity may be related with the changes of hydrogen bonding interactions between the side chain of residue at the four positions (47, 89, 94, and 196) and the substrate sugars.