Cyanobacteria have attracted significant interest as a platform for renewable production of fuel and feedstock chemicals from abundant atmospheric carbon dioxide by way of photosynthesis. While great strides have been made in developing this technology in freshwater cyanobacteria, logistical issues remain in scale-up. Use of the cyanobacterium Synechococcus sp. PCC 7002 (7002) as a chemical production chassis could address a number of these issues given the higher tolerance to salt, light, and heat as well as the fast growth rate of 7002 in comparison to traditional model cyanobacteria such as Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803. However, despite growing interest, the development of genetic engineering tools for 7002 continues to lag behind those available for model cyanobacterial strains. In this work we demonstrate the systematic development of a 7002 production strain for the feedstock chemical 2,3-butanediol (23BD). We expand the range of tools available for use in 7002 by identifying and utilizing new integration sites for homologous recombination, demonstrating the inducibility of theophylline riboswitches, and screening a set of isopropyl β-d-1-thiogalactopyranoside (IPTG) inducible promoters. We then demonstrate improvements of 23BD production with the systematic screening of different conditions including: operon arrangement and copy number, light strength, inducer concentration, cell density at the time of induction, and nutrient concentration. Final production tests yielded titers of 1.6 g/L 23BD after 16 days at a rate of 100 mg/L/day. This work represents great strides in the development of 7002 as an industrially relevant production host.Keywords: 2,3-butanediol; cyanobacteria; marine cyanobacteria; metabolic engineering;

Cyanobacteria have attracted much attention as a means to directly recycle carbon dioxide into valuable chemicals that are currently produced from petroleum. However, the titers and productivities achieved are still far below the level required in industry. To make a more industrially applicable production scheme, glycerol, a byproduct of biodiesel production, can be used as an additional carbon source for photomixotrophic chemical production. Glycerol is an ideal candidate due to its availability and low cost. In this study, we found that a heterologous glycerol respiratory pathway enabled Synechococcus elongatus PCC 7942 to utilize extracellular glycerol. The engineered strain produced 761 mg/L of 2,3-butanediol in 48 h with a 290% increase over the control strain under continuous light conditions. Glycerol supplementation also allowed for continuous cell growth and 2,3-butanediol production in diurnal light conditions. These results highlight the potential of glycerol as an additional carbon source for photomixotrophic chemical production in cyanobacteria.Keywords: 2,3-butanediol; cyanobacteria; glycerol metabolism; metabolic engineering; obligate photoautotroph;

Cyanobacteria have gained popularity among the metabolic engineering community as a tractable photosynthetic host for renewable chemical production. However, though a number of successfully engineered production systems have been reported, long-term genetic stability remains an issue for cyanobacterial systems. The genetic engineering toolbox for cyanobacteria is largely lacking inducible systems for expression control. The characterization of tight regulation systems for use in cyanobacteria may help to alleviate this problem. In this work we explore the function of the IPTG inducible promoter PLlacO1 in the model cyanobacterium Synechococcus elongatus PCC 7942 as well as the effect of gene order within an operon on pathway expression. According to our experiments, PLlacO1 functions well as an inducible promoter in S. elongatus. Additionally, we found that gene order within an operon can strongly influence control of expression of each gene.Keywords: 2,3-butanediol; cyanobacteria; lac promoter

Global energy and environmental concerns have driven the development of biological chemical production from renewable sources. Biological processes using microorganisms are efficient and have been traditionally utilized to convert biomass (i.e., glucose) to useful chemicals such as amino acids. To produce desired fuels and chemicals with high yield and rate, metabolic pathways have been enhanced and expanded with metabolic engineering and synthetic biology approaches. 2-Keto acids, which are key intermediates in amino acid biosynthesis, can be converted to a wide range of chemicals. 2-Keto acid pathways were engineered in previous research efforts and these studies demonstrated that 2-keto acid pathways have high potential for novel metabolic routes with high productivity. In this review, we discuss recently developed 2-keto acid-based pathways.

Photosynthetic chemical production in cyanobacteria is a promising technology for renewable energy, CO2 mitigation, and fossil fuel replacement. Metabolic engineering has enabled a direct biosynthetic process from CO2 fixation to chemical feedstocks and biofuels, without requiring costly production, storage, and breakdown of cellulose or sugars. However, direct production technology is challenged by a need to achieve high-carbon partitioning to products in order to be competitive. This review discusses principles for the design of biosynthetic pathways in cyanobacteria and describes recent advances in relevant genetic tools. This field is at a critical juncture in assessing the strength and feasibility of carbon partitioning. To address this, we have included the stoichiometry of reducing equivalents and carbon conservation for heterologous pathways, and a method for calculating product yields against a theoretical maximum.

Conversion of CO2 for the synthesis of chemicals by photosynthetic organisms is an attractive target for establishing independence from fossil reserves. However, synthetic pathway construction in cyanobacteria is still in its infancy compared with model fermentative organisms. Here we systematically developed the 2,3-butanediol (23BD) biosynthetic pathway in Synechococcus elongatus PCC7942 as a model system to establish design methods for efficient exogenous chemical production in cyanobacteria. We identified 23BD as a target chemical with low host toxicity, and designed an oxygen-insensitive, cofactor-matched biosynthetic pathway coupled with irreversible enzymatic steps to create a driving force toward the target. Production of 23BD from CO2 reached 2.38 g/L, which is a significant increase for chemical production from exogenous pathways in cyanobacteria. This work demonstrates that developing strong design methods can continue to increase chemical production in cyanobacteria.

•Metabolic engineering of cyanobacteria for biofuel and bulk chemical production.•Photoheterotrophic production is improving titers in light and dark conditions.•Terpenoid biosynthesis is expanding range of biofuels from cyanobacteria.•Biodegradable plastic feedstocks and carbohydrate production are valuable and promising atmospheric carbon sinks.Rising levels of atmospheric CO2 are contributing to the global greenhouse effect. Large scale use of atmospheric CO2 may be a sustainable and renewable means of chemical and liquid fuel production to mitigate global climate change. Photosynthetic organisms are an ideal platform for efficient, natural CO2 conversion to a broad range of chemicals. Cyanobacteria are especially attractive for these purposes, due to their genetic malleability and relatively fast growth rate. Recent years have yielded a range of work in the metabolic engineering of cyanobacteria and have led to greater knowledge of the host metabolism. Understanding of endogenous and heterologous carbon regulation mechanisms leads to the expansion of productive capacity and chemical variety. This review discusses the recent progress in metabolic engineering of cyanobacteria for biofuel and bulk chemical production since 2014.

•We examine biofuel and biofuel precursor production by photosynthetic microorganisms.•We review bioenergy production (mainly hydrogen) by photosynthetic microorganisms.•We discuss pigment and antioxidant production by algae and cyanobacteriaNational interest and environmental advocates encourage alternatives to petroleum-based products. Besides biofuels, many other valuable chemicals used in every-day life are petroleum derivatives or require petroleum for their production. A plausible alternative to production using petroleum for chemical production is to harvest the abundant carbon dioxide resources in the environment to produce valuable hydrocarbons. Currently, efforts are being made to utilize a natural biological system, photosynthetic microorganisms, to perform this task. Photosynthetic microorganisms are attractive to use for biochemical production because they utilize economical resources for survival: sunlight and carbon dioxide. This review examines the various compounds produced by photosynthetic microorganisms.Download high-res image (152KB)Download full-size image