Succinate has been recognized as an important platform chemical that can be produced from biomass. While a number of organisms are capable of succinate production naturally, this review focuses on the engineering of Escherichia coli for production of the four-carbon dicarboxylic acid. Important features of a succinate production system are to achieve optimal balance of reducing equivalents generated by consumption of the feedstock, while maximizing the amount of carbon that is channeled to the product. Aerobic and anaerobic production strains have been developed and applied to production from glucose as well as other abundant carbon sources. Metabolic engineering methods and strain evolution have been used and supplemented by the recent application of systems biology and in silico modeling tools to construct optimal production strains. The metabolic capacity of the production strain, as well as the requirement for efficient recovery of succinate and the reliability of the performance under scale-up are important in the overall process. The costs of the overall biorefinery compatible process will determine the economical commercialization of succinate and its impact in larger chemical markets.

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E. coli strains HL2765 and HL27659k harboring pRU600 and pKK313 were examined for succinate production under aerobic conditions using galactose, sucrose, raffinose, stachyose, and mixtures of these sugars extracted from soybean meal and soy solubles. HL2765(pKK313)(pRU600) and HL27659k(pKK313)(pRU600) consumed 87 mM and 98 mM hexose of soybean meal extract and produced 83 mM and 95 mM succinate, respectively. While using soy solubles extract, HL2765(pKK313)(pRU600) and HL27659k(pKK313)(pRU600) consumed 160 mM and 187 mM hexose and produced 158 mM and 183 mM succinate, respectively. Succinate yield of HL2765(pKK313)(pRU600) was low as compared to that of HL27659k(pKK313)(pRU600) while using acid hydrolysate of soybean meal or soy solubles extracts. Maximum succinate production of 312 mM with a molar yield of 0.82 mol/mol hexose was obtained using soy solubles hydrolysate by HL27659k(pKK313)(pRU600). This study demonstrated the use of soluble carbohydrates of the renewable feedstock, soybean as an inexpensive carbon source to produce succinate by fermentation.

Clostridium acetobutylicum is a natural producer of butanol, butyrate, acetone and ethanol. The pattern of metabolites reflects the partitioning of redox equivalents between hydrogen and carbon metabolites. Here the exogenous genes of ferredoxin-NAD(P) oxidoreductase (FdNR) and trans-enoyl-coenzyme reductase (TER) are introduced to three different Clostridium acetobutylicum strains to investigate the distribution of redox equivalents and butanol productivity. The FdNR improves NAD(P)H availability by capturing reducing power from ferredoxin. A butanol production of 9.01 g/L (36.9% higher than the control), and the highest ratios of butanol/acetate (7.02) and C/C (3.17) derived metabolites were obtained in the C acetobutylicum buk strain expressing FdNR. While the TER functions as an NAD(P)H oxidase, butanol production was decreased in the C. acetobutylicum strains containing TER. The results illustrate that metabolic flux can be significantly changed and directed into butanol or butyrate due to enhancement of NAD(P)H availability by controlling electron flow through the ferredoxin node.

26The review describes efforts toward metabolic engineering of production of organic acids. One aspect of 27 the strategy involves the generation of an appropriate amount and type of reduced cofactor needed for the 28 designed pathway. The ability to capture reducing power in the proper form, NADH or NADPH for the 29 biosynthetic reactions leading to the organic acid, requires specific attention in designing the host and also 30 depends on the feedstock used and cell energetic requirements for efficient metabolism during production. 31Recent work on the formation and commercial uses of a number of small mono and diacids is discussed 32 with redox differences, major biosynthetic precursors and engineering strategies outlined. Specific 33 attention is given to those acids that are used in balancing cell redox or providing reduction equivalents for 34 the cell, such as formate, which can be used in conjunction with metabolic engineering of other products to 35 improve yields. Since a number of widely studied acids derived from oxaloacetate as an important 36 precursor, several of these acids are covered with the general strategies and particular components 37 summarized, including succinate, fumarate and malate. Since malate and fumarate are less reduced than 38