Project Summary
Formula provides nutrition to infants as a partial or complete substitute for human milk for working mothers or in
situations like lactation failure or poor maternal health. The heavy reliance on infant formula in the U.S. is
reflected by a National Immunization Survey which found that only ~25% of infants (< 6 months) were exclusively
breastfed between 2018-19, a number that was even smaller in rural areas and low-income families. The fat
composition of formulas serves a vital role in replacing human milk fat (HMF), which provides >50% of energy
to the infant during early lactation. However, derived from vegetable oils, the fat in commercial formulas often
lacks the unique fatty acid composition and lipid structure of HMF, resulting in reduced dietary benefits and
poorer health outcomes for infants. The distinct features of HMF include but are not limited to 1) a substantial
portion of medium-chain (C8:0-C14:0) fatty acids and 2) the prevalence at the sn-2 position of palmitic acid (a
C16:0 fatty acid) on triacylglycerols (TAGs). Medium-chain fatty acids are desirable as a quick source of energy
for infants because of their higher absorption rates compared to longer fatty acids. C16:0 esterification at the sn-
2 position prevents its release during digestion by the sn-1,3 lipase. This is important to avoid 16:0 precipitation,
which chelates essential calcium ions and causes hard stools. Present methods for correcting fatty acid
composition and TAG regioisomeric structure in formula are expensive and because of their reliance on palm
oil, undesirable from an environmental perspective. This project addresses these challenges through the
development of an HMF substitute in metabolically engineered algae: the goal is to generate a healthy,
environmentally friendly, and low-cost product for US consumption. Auxenochlorella protothecoides (A.pro)
presents an ideal platform for this goal because of 1) its exceptional biomass and oil accumulating capabilities
compared to traditional oil crops, 2) convenient genetic and genome modification tools to facilitate metabolic
engineering, and 3) the potential for producing the product at commercial scale. In this context, a fatty acid
substrate pool resembling the fatty acid composition of HMF will be built by heterologous expression of chain
length-determining fatty acyl-ACP thioesterases with desired substrate specificities (e.g., C8:0-C14:0) in A.pro,
followed by GC-MS product analysis for assessment (Aim 1). To achieve TAG products with the correct regio-
selectivity (C16:0 at sn-2), the C16:0-specific lysophosphatidic acid acyltransferase will be introduced, followed
by stereochemical analysis of TAGs by sn-1,3 lipases treatment and GC-MS analysis of the isolated
monoacylglycerols (Aim 2). Finally, the endogenous polycistronic gene expression system, discovered recently
by the sponsor, will be exploited for simultaneous expression of rate-limiting enzymes in fatty acid biosynthesis,
which will streamline the engineering process and enable more efficient production of HMF in A.pro (Aim 3). In
summary, this research will explore the feasibility of producing HMF in oleaginous green algae for infant formula
and also pave the way for developing a promising synthetic biology approach for other bioproducts in A.pro.